Anatomical Changes in Peach Leaves Infected by Taphrina Deformans (Berk.) Tul

ECOLOGIA BALKANICA 2014, Vol. 5, Special Edition April 2014 pp. 101-106

Anatomical Changes in Peach Leaves Infected by Taphrina deformans (Berk.) Tul.

Ivanka B. Semerdjieva*¹, Neshka G. Piperkova², Mariya V. Zarkova³, Lyubka H. Koleva-Valkova

1 - Department of Botany and Agrometeorology, ⁴ Agricultural University – Plovdiv, Plovdiv, BULGARIA 2 - Department of Phytopathology, Agricultural University – Plovdiv, Plovdiv, BULGARIA 3 - Department of Accountancy and Statistics, Agricultural University – Plovdiv, Plovdiv, BULGARIA 4 -Department of Plant physiology and biochemistry; Agricultural University – Plovdiv, Plovdiv, BULGARIA * Corresponding author: [email protected]

Abstract. Light microscope study of Prunus persica (L.) Batsch. (Fayette cultivar) leaf anatomical structure, naturally infected by Taphrina deformans (Berk.) Tul. has been conducted. In the infected leaves histological changes were observed such as increase of the total thickness of the mesophyll and a loss of its differentiation to palisade and spongy parenchyma. An increase in the size of the upper epidermis was established as a result of fungus localization. The results were supported by morphometric and statistical analysis.

Key words: leaf curl, Prunus persica; Taphrina deformans; leaf anatomy.

Introduction GARGIULO, 1972; SYROP, 1975; HUANG et al., Peach leaf curl caused by Taphrina 1993; ADEKUNLE et al., 2005. The hormone deformans (Berk.) Tul. is a disease widely imbalance leading to hypertrophy and spread and potentially harmful in cool and hyperplasia in the infected leaves is in a humid spring seasons. Morphological traits causal relationship with the changes on a are related to leaf deformation and changes physiological and structural level (SZIRAKI et in pigmentation (chlorotic and anthocyanic al. 1975; YMADA et al. 1990; TSAVKELOVA et coloration) of leaves, shoots and al. 2006). occasionally of fruits (Fig. 1). They are due Using light and electron microscopic to deep physiological alterations leading to observations, SYROP (1975) and HUANG et al. premature aging and leaf abscission. Such (1993) detected similar changes in almond are the abnormalities in the leaf gas- and peach leaves infected by Taphrina exchange, pigment and water content, etc. deformans. They pointed out that accelerated (RAGGY, 1966; MONTALBINI & BUONAURIO, cell division was the first response of the 1986; RAGGY, 1995; PIPERKOVA & VASILEV, host. Changes in the palisade tissue, 2000). Changes in the structure of the leaf deformation and decreased size of the blade and of the cell shape and structure chloroplasts in the cells surrounded by the were described in the papers of MARTE & pathogen hyphae were found out, followed

Anatomical Changes in Peach Leaves Infected by Taphrina deformans (Berk.) Tul. by the development of chlorosis. BASSI et al. pumilio leaves infected by Taphrina (1984), GIORDANI et al. (2012) proved in their entomospora. studies that the mesophyll layers could not Similar studies have not been carried be well-defined in the leaves infected by out in Bulgaria. In the present investigation Taphrina deformans. Similar results were also the authors offer their vision on the established in the investigation carried out structural and anatomical changes detected by HANSEN et al. (2007) on Nothofagus in peach leaves of Fayette cultivar.

Fig. 1. Symptoms of naturally infected by Taphrina deformans leaves of Prunus persica (L.) Batsch.

Material and methods and post fixed with 1.3% (w/v) OsO4 in the The samples for the analyses were same buffer. Infiltration and embedding prepared from peach leaves of Fayette were performed using Durcupan ACM cultivar (Prunus persica (L.) Batsch), (Fluka, Sigma – Aldrich). Semi-thin section naturally infected by Taphrina deformans, as (1 -2 µm), cut from the Durcupan – well as healthy leaves (control), collected at embedded material with glass knives, were the end of April 2011 in the region of mounted on glass slides, stained with Plovdiv. fuchsin and methylene blue and examined Both variants were studied following under a NU light microscope (Zeiss, Jena, the standard methods of comparative Germany). Metric characteristics of the anatomy (NIKOLOV & DASKALOV, 1966; studied samples included: thickness of the METCALFE & CHALK, 1979). The cross- adaxial (ad) and abaxial epidermis (ab) in sections of fresh materials were studied µm, total thickness of the mesophyll in µm, from the middle part of the lamina. Semi- thickness of the palisade and spongy stable microscopic preparations were made. mesophyll layers in µm. 30 measurements of Amplival microscope was used for the light each characteristic were obtained. study. Measurements were made with an The statistical analysis of empiric data eyepiece micrometer (10х) and the pictures includes point and interval estimation of were taken with a light digital microscope parameters: mean on each variable (metric Motic DMBA-210. statistic), and difference between means for Part of leaves of fully development infected and control leaves, at a 95 % infected and healthy leaves (control) were confidence level. Null Hypothesis for no processed following the protocol, described effect of infection is proved by a t-test for by DONCHEVA et al. (2001). Briefly, the independent samples at a significance level samples were fixed in 5% (v/v) α = 0,05. Programs were used for data glutaraldehyde in 0.1M Na – cacodilate processing STATISTICA for Windows buffer (pH 7) for 2 h at room temperature (STATSOFT INC., 2007).

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Ivanka B. Semerdjieva, Neshka G. Piperkova, Mariya V. Zarkova, Lyubka H. Koleva-Valkova Results and Discussion dicotyledonous plants, leaves of which have Studies on the anatomical structure of a flat blade and are horizontally arranged. the leaves are of great importance for The palisade parenchyma is structured by explaining the adaptive responses of the oval-cylindrical cells arranged in two rows, plants to ecological or pathogenic stress. with well-developed protoplasma organelles By means of light microscope analysis and small intercellular spaces for free gas- of cross-sections samples of healthy peach exchange, the average thickness being 50.58 leaves (control), it was found out that the µm (Table 1). The cells of the spongy epidermal tissue was formed by a single cell parenchyma are irregular in shape, arranged layer. The mean height of the adaxial in several rows, with large intercellular epidermis (ad) was 14.33 µm, and of the spaces. Its average thickness is 52.41 µm abaxial one (ab) – 12.50 µm (Table 1). The (Fig. 2; 3, Table 1). Vascular bundles are mesophyll was of a clearly defined palisade located at the border between palisade and and spongy parenchyma, i.e. dorsoventral spongy parenchyma. type. That structure is typical of most

Fig. 2. General view of the transverse section of healthy leaves of Prunus persica (L.) Batsch. Fayette cultivar, LM at magnification X 100 (ad ep – upper epidermis; ab ep – lower epidermis; PP – palisade parenchyma; SP – spongy parenchyma).

Fig. 3. Semi-thin section of healthy leaves of Prunus persica (L.) Batsch. Fayette cultivar, LM at magnification X 400.

As it is known, the parasitizing hyphae 2012). They cause histological changes like of Taphrina deformans develop in the hypertrophy and hyperplasia, provoked by intercellular spaces of the mesophyll and biologically active substances (SZIRAKI et al., the epidermis (SYROP, 1975; GIORDANI et al. 1975; YMADA et al., 1990). Statistically

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Anatomical Changes in Peach Leaves Infected by Taphrina deformans (Berk.) Tul. significant histological changes in the leaf processes of hypertrophy and hyperplasia, structure were found out in the samples provoked by the pathogen, the shape of the prepared by infected plants. They were cells from the palisade layer turned from detected in the height of the adaxial (ad) cylindrical oval to isodiametric. Both, large and abaxial epidermis (ab), in the thickness hypertrophied, strongly vacuolated cells, of the leaf lamina, in the shape and size of with organelles located close to the cell wall, the mesophyll cells. The adaxial epidermis with small intercellular spaces were height increased by about 45% compared to observed, together with cells twice or thrice the control, i.e. by 6.50 µm in average, while smaller in size (Fig. 4.). the abaxial decreased from 12.50 to 10.25 The morphometric analysis of the cross- µm, i.e. by about 22% (Table 1). The results section of the control and of the infected obtained about the abaxial epidermis height leaves confirmed the histological changes did not agree with those provided by (Table 1). The total average thickness of the HANSEN et al. (2007) in their studies of mesophyll in the first variant was 120.92 µm, Nothofagus pumilio infected by Taphrina while in the infected leaves it reached up to entomospora. The reason for that discrepancy 197.50 µm. The registered difference of 76.58 in the obtained results is due to the fact that µm is statistically significant. the parasitizing mycelium of Taphrina The established changes in the structure deformans at the end of the pathological of the leaf lamina correlated with some process develops subcuticularly, between changed parameters of the physiological the adaxial epidermis cells, while the state of the peach leaves infected by Taphrina mycelium of Taphrina entomospora develops deformans, to which Fayette cultivar is on the abaxial epidermis. Accordingly, the susceptible. The chlorophyll content asci are formed on the upper leaf surface in decreased by 22-29%, the photosynthesis peach and on the lower one in Nothofagus rate was inhibited in the range of 17 to 45% pumilio. It could be admitted that thickening and breathing increased repeatedly of the epidermal layer, spores of the (PIPERKOVA & VASILEV, 2000). That pathogen are bursting through, is a confirmed the fact that the histological protective mechanism of the host. changes established in the present study by Disrupted mesophyll differentiation light microscopy, as well as the changes in was established in the areas with peach cultivars showing different resistance parasitizing mycelium, i.e. the leaf lamina to the pathogen, found out by GIORDANI et changed from dorsoventral to isolateral al. (2012), are causally related to the (equifacial) (Fig. 4.). In result of the disturbed metabolism in the infected leaves.

Fig. 4. General view of transverse section of infected by Taphrina deformans leaves of Prunus persica (L.) Batsch. LM at magnification X 100 (ad ep – upper epidermis; ab ep – lower epidermis; spongy parenchyma.

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Ivanka B. Semerdjieva, Neshka G. Piperkova, Mariya V. Zarkova, Lyubka H. Koleva-Valkova

Fig. 5. Semi-thin section of infected by Taphrina deformans leaves of Prunus persica (L.) Batsch. LM at magnification X 400 (M – mycelium).

Table 1. Morphometric parameters of healthy (control) and infected by Taphrina deformans (Berk.)Tul. leaves of Prunus persica(L.) Batsch.

95 % Confidence Interval Parameters Standard Difference Mean Deviation (SD) between Means Upper epidermis (ad), control 14.33 ± 0.65 3.9108 6.50 ± 0.87* thickness/μm infected 20.83 ± 0.62 21.3247 Lower epidermis (ab), control 12.50 ± 0.60 1.7287 -2.25 ± 0.84* thickness/μm infected 10.25 ± 0.62 1.6522 control 120.92 ± 1.46 1.6082 Mesophyll thickness, μm 76.58 ± 8.07* infected 197.50 ± 7.96 1.6544 Palisade mesophyll, control 50.58 ± 1.09 - thickness/μm 2.9127 Spongy mesophyll, control 52.42 ± 1.44 - thickness/μm 3.8553 *statistically significant difference at α = 0.05

Conclusion decrease of the abaxial epidermis height by In result of the study carried out with 22%, which was caused by the Taphrina light microscopy on healthy and naturally deformans asci formed on the upper surface infected by Taphrina deformans peach leaves of the peach leaves. The established changes (Prunus persica (L.) Batsch.) of Fayette were statistically significant. Structural cultivar, severe histological changes were differences in the infected leaves were established. The leaf structure changed directly related to the changed physiological considerably from dorsoventral to isolateral. state of the plant. Under the influence of the processes of hypertrophy and hyperplasia, the plant cells References located around the fungal hyphae, were strongly vacuolized, they increased in size ADEKUNLE A.A., N.U. UMA, A. M. OLEAH. and acquired an isodiametric shape. The 2005. Studies on Coloured Leaf Spot morphometric analysis confirmed the Disease of Alchornea cordifolia Caused increase of the adaxial epidermis height by by Taphrina deformans. - Plant Pathology 45% compared to the control and the Journal, 4 (2): 150-156.

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Anatomical Changes in Peach Leaves Infected by Taphrina deformans (Berk.) Tul.

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